| Rheumatoid arthritis(RA)is a systemic autoimmune disease,which is characterized by inflammatory changes in the synovial tissue of the joints,hyperplasia of synovial tissue and vascular formation,and gradually the emergence of joint cartilage damage and bone destruction,which eventually leads to joint deformities and loss of function.The cause of RA has not been fully elucidated.Previous studies have shown that both genetic and epigenetic factors can influence the occurrence of RA.As one of the most abundant RNA modifications,N6-methyladenosine(m6A)modification is necessary for the biogenesis and function of RNA,and its modification abnormalities are associated with various diseasrs.However,the role of m6A methylation modification in RA is unclear.A large number of studies have shown that m6A methylation modification is involved in regulating the biological functions of a variety of immune cells,such as peripheral blood mononuclear cells(PBMC),macrophages,dendritic cells,T cells,etc.Given that these immune cells and fibroblast-like synoviocytes(FLS)play a key role in the pathological process of RA,we speculate that the potential role of m6A methylation modification in RA may be achieved by regulating the biological functions of different immune cells and FLS,but its specific role and mechanism are not clear.In addition,drugs developed based on m6A methylation modifications are very rare,and although current inhibitors of m6A modification of demethylase fat and obesity-associated proteins have been developed,their effects have only been verified by in vitro and in vitro experiments,and their effectiveness and safety remain to be evaluated.What’s more,no drugs based on m6A methylation modifications for the treatment of RA have been developed.Based on the above questions,this paper mainly includes the following three parts:Part Ⅰ:Study of the role and mechanism of RNA m6A-modified demethylase ALKBH5 in FLS under hypoxia condition.Methods:(1)The synovial tissues of the knee joint of RA patients and osteoarthritis patients(OA)were collected.Then,immunohistochemistry(IHC),RT-PCR and Western blot methods were used to detect the gene and protein levels of hypoxia inducible factor(HIF)-1α,HIF-2α and ALKB homologue 5(ALKBH5)in tissues,and the role of HIF-1α,HIF-2α and ALKBH5 in the pathological process of RA was preliminarily explored;(2)Collagen-induced arthritis(CIA)rat model was constructed,arthritis scoring(AI)was used to evaluate the incidence of rats,hematoxylin-eosin(HE)staining,crocus O-solid green staining and Micro-CT were used to evaluate the joint pathology and bone destruction of CIA rat model,and IHC,RT-PCR and Western blot methods were applied to detect gene and protein levels of HIF-1α,HIF-2α and ALKBH5 in different tissues,clarifying their role in CIA animal model;(3)① FLSs were placed in normal oxygen and hypoxia environments for culture,the effect of hypoxia environment on FLS proliferation was detected by CCK-8 method,the effect of hypoxia environment on FLS migration and invasion were evaluated by cell scratch,transwell and TRITC phalloidin staining.The effects of hypoxia on HIF-1α,HIF-2α and ALKBH5 genes and protein levels in FLS were detected by RT-PCR and Western blot methods,and the effects of hypoxia environment on TNF-α,IL-1β and IL-6 mRNA in FLS were detected by RT-PCR method.②In hypoxic and normal oxygen environments,the effects of overexpression/knockdown of ALKBH5 on cell migration and invasion were detected by cell scratch experiment,transwell experiment and TRITC phalloidin staining.The effects of overexpression/knockdown of ALKBH5 on TNF-α,IL-1β and IL-6 mRNA in FLS were detected by RT-PCR method.③ In hypoxic environment,ALKBH5 was knocked down in FLS and cells were collected for MeRIP-seq detection and analysis,MeRIP-qPCR,RT-PCR and Western blot methods were used to screen for potential target genes downstream of ALKBH5.④ The relationship between ALKBH5 and the potential target gene CH25H in hypoxic environments was verified by RT-PCR,Western blot,RNA stability experiment,transwell experiment,TRITC phalloidin staining,gene overexpression and gene knockdown.⑤ Data from the GEO public database were analyzed and methods such as RT-PCR,IHC,immunofluorescence and correlation analysis were used to analyze and detect the expression of CH25H in RA synovial tissue,clarifying the role of CH25H in the pathological process of RA.Results:(1)Compared with OA patient tissues,the gene and protein levels of HIF-1α,HIF-2α and ALKBH5 in the synovial tissues of RA patients were significantly upregulated;(2)The onset of the disease began on the 3rd to 4th day after the second immunization,and entered the peak of the disease on the 6th to 8th days after the second immunization;typical photos of the appearance of the rat’s hind paws showed that the hind paws of rats in CIA model were severe swelling;compared with the normal group,serious pathological damage and bone destruction were appeared in the ankle joints of CIA animals;compared with the normal group,the gene and protein expression of HIF-1α,HIF-2α and ALKBH5 in the ankle and knee synovial tissues of the CIA animals were upregulated;(3)①Compared with the normal oxygen environment,the hypoxic environment can promote the proliferation,migration and invasion of FLS,promote the gene and protein expression of HIF-1α,HIF-2α and ALKBH5 in FLS,as well as promoting the expression of TNF-α,IL-1β and IL-6 mRNA in FLS.② In hypoxic environment,overexpression of ALKBH5 can promote the migration,invasion and expression of TNF-α,IL-1β and IL-6 mRNA of FLS,while knocking down ALKBH5 can inhibit the migration,invasion and expression of TNF-α,IL-1β and IL-6 mRNA.③ From the MeRIP-seq results,49 genes with significant changes in m6A methylation modification and transcription level were found,the G protein-coupled receptor-binding proteins alpha 14(GNA14),cholesterol 25-hydroxylase(CH25H),and B-lymphocytoma 2(BCL2),which have relatively high transcriptome expression and higher variations in multiples,were screened and validated.The results showed that after knocking down ALKBH5,the m6A modification levels of GNA14,CH25H and BCL2 were increased.The results of RT-PCR and Western blot showed that after knocking down ALKBH5,the BCL mRNA and protein levels did not change significantly,the GNA14 and CH25H mRNA and protein expression were significantly downregulated,and the CH25H downregulation was more obvious,so CH25H was determined to be a potential target gene downstream of ALKBH5.④In hypoxic environment,ALKBH5 expression can affect the stability of CH25H mRNA;knocking down CH25H can inhibit FLS migration and invasion,downregulate the expression of TNF-α,IL-1β,IL-6 mRNA in FLS;overexpression of CH25H can promote FLS migration and invasion,and upregulate the expression of TNF-α,IL-1β,IL-6 mRNA in FLS.And CH25H overexpression reverses the weakening of FLS migration and invasion ability and decreased expression of inflammatory factors caused by low expression of ALKBH5.⑤In database of GSE55457,GSE55235,compared with OA patients,the level of CH25H in the synovial tissue of RA patients was significantly upregulated;In GSE36700 database,CH25H level in synovial tissue in RA patients was also higher than that in OA patient,but there was no significant difference;The RT-PCR result showed that the mRNA expression of CH25H in the synovial tissue of RA patients was significantly increased compared with patients in the OA group;The IHC result showed that CH25H was upregulated in the synovial tissue of RA patients,and immunofluorescence showed that CH25H and ALKBH5 were upregulated in the synovial tissue of RA patients compared with the OA group;The correlation analysis showed that the mRNA level of CH25H was negatively correlated with the m6A level and positively correlated with the ALKBH5 mRNA level.These results indicates that the upregulation of ALKBH5 and CH25H in synovial tissue is directly related to the pathological process of RA.Part Ⅱ:Study of the role of RNA m6A methylation modification in RA PBMC.Methods:(1)PBMCs of RA patients were collected,MeRIP-seq and RNA-seq were performed,and the map of m6A methylation modification of RA PBMC was ploted;(2)Differential methylation modified genes were analyzed based on MeRIP-seq results,and the biological functions and signaling pathways involved in differential methylation modified genes were analyzed;(3)Differential genes were analyzed according to RNA-seq results,and the biological functions and signaling pathways involved in differential genes were analyzed;(4)The combined analysis of MeRIP-seq and RNA-seq was carried out to analyze genes with differential methylation modification and transcription levels,and the biological functions and pathways involved in these differential genes were analyzed to clarify the key role of m6A methylation modification in RA PBMC.Results:(1)m6A methylation modification pattern of RA PBMC was plotted;(2)35 genes with significant differences in m6A methylation modification and mRNA levels were screened;(3)These genes with significant differences in methylation modification and mRNA levels participated in multiple biological functions and signaling pathways,and played an important role in RA.Part Ⅲ:Based on the research content of Part Ⅱ,a new target for triptolide(TP)treatment of RA was excavated.Methods:(1)Based on the results of MeRIP-seq and RNA-seq of RA PBMC,combined with bioinformatics analysis,new targets of TP treatment of RA were excavated;(2)The data of synovial tissue in different sets of GEO database were retrieved to analyze the expression of the target genes;(3)Molecular docking and RT-PCR methods were used to preliminarily verify the new targets of TP therapy for RA.Results:(1)There is a close network connection between the target proteins of TP and the RA differential genes analyzed in the second part,especially there are more network connections between genes such as insulin-like growth factor 2 binding protein 3(IGF2BP3),TUBB2A,DYNC1I1,FOSL1 and the target proteins of TP;(2)In the 4 datasets of the GEO database,the expression of IGF2BP3 in synovial tissue was significantly upregulated compared with normal synovial tissue and OA synovial tissue,and its expression trend was consistent with that in PBMC,which suggested that expression of IGF2BP3 in tissues and cells was stable;(3)The molecular docking results showed that the affinity fraction between TP and IGF2BP3 was-8.6 kcal/mol,indicating that TP and IGF2BP3 had strong binding activity.The RT-PCR results showed that TP can reduce the expression of IGF2BP3 in PBMC and synovial fibroblast line MH7A,suggesting that IGF2BP3 is a potential new target for TP treatment of RA,but its in-depth mechanism of action will continue to be explored in follow-up studies.Conclusions:(1)The HIF1α/2α-ALKBH5-CH25H pathway plays a key role in the pathological process of RA,which may be one of the key pathways for RA FLS migration,invasion and inflammation;(2)The m6A methylation modified genes in RA PBMC is closely related to disease,and m6A methylation modification plays an important role in RA PBMC;(3)TP may play a therapeutic role in RA by targeting the m6A methylated modified binding protein IGF2BP3. |
